Monoterpene Indole Alkaloids from the Aerial Parts of Rhazya stricta Induce Cytotoxicity and Apoptosis in Human Adenocarcinoma Cells

Chromatographic investigation of the aerial parts of the Rhazya stricta (Apocynaceae) resulted in the isolation of two new monoterpene indole alkaloids, 6-nor-antirhine-N1-methyl (1) and razyamide (2), along with six known compounds, eburenine (3), epi-rhazyaminine (4), rhazizine (5), 20-epi-sitsirikine (6), antirhine (7), and 16-epi-stemmadenine-N-oxide (8). The chemical structures were established by various spectroscopic experiments. Compounds 1–8 exhibited cytotoxic effects against three cancer cells with IC50 values ranging between 5.1 ± 0.10 and 93.2 ± 9.73 µM against MCF-7; 5.1 ± 0.28 and 290.2 ± 7.50 µM against HepG2, and 3.1 ± 0.17 and 55.7 ± 4.29 µM against HeLa cells. Compound 2 showed the most potent cytotoxic effect against all cancer cell lines (MCF-7, HepG2 and HeLa with IC50 values = 5.1 ± 0.10, 5.1 ± 0.28, and 3.1 ± 0.17 µM, respectively). Furthermore, compound 2 revealed a significant increase in the apoptotic cell population of MCF-7, HepG2, and HeLa cells, with 31.4 ± 0.2%, 29.2 ± 0.5%, and 34.9 ± 0.6%, respectively. Compound 2 decreased the percentage of the phagocytic pathway on HepG2 cells by 15.0 ± 0.1%. These findings can explain the antiproliferative effect of compound 2.


Introduction
Cancer is an uncontrollable growth of cells, it could be generated or disseminated in different organs. It represents a major world health problem and increases the global mortality rate. Additionally, it is identified as the second cause of death after cardiovascular diseases [1]. In 2018, around 10 million people died from cancers [2]. The most common cancers were diagnosed in breast, lung, colon and rectum, prostate, skin (non-melanoma), and stomach with the number of cases, 2.26, 2.21, 1.93, 1.41, 1.20, and 1.09 million, whereas
The 2D spectra of compound 1 including the 1 H-1 H COSY, HSQC, and HMBC spectra showed great similarity to a previously isolated alkaloid known as antirhine) [24]. However, a deep comparison between both alkaloids revealed the absence of a methylene function in the case of 1 (CH2-6) and instead of the appearance of a methyl function. The location of the methyl function was deduced from the down-field absorptions in both 1 H and 13 C NMR and also from the correlation between these methyl protons and the carbons be linked to the nitrogen atom of the indole ring. From the comparison between the above Compound 1 was obtained as a yellow oily substance of molecular formula C 19 H 24 N 2 O, as established by HR-EIS-MS. The presence of N-containing structure was evidenced from the orange color developed upon spraying with Dragendorff's reagent; however, the presence of indole moiety has been evidenced from four absorption bands at 227, 276, 284, and 291 nm observed in the UV spectrum. Absorption bands at 3250, 3067, 1599, and 1456 cm −1 in the IR spectrum indicated the presence of OH, CH-, and a benzene ring, respectively. Four aromatic proton signals appeared doublets (d), doublets of doublets of doublets (ddd), doublets of doublets of doublets (ddd), and doublets (d) in the 1 H nmR spectrum resonating at δ H 7.38 (1H, d, J = 7.8 Hz), 6.96 (1H, ddd, J = 7.8, 7.8,1.2 Hz), 7.01 (1H, ddd, J = 7.8, 7.8, 1.2 Hz), and 7.30 (1H, d, J = 7.8 Hz), respectively, pointed out to the presence 1,2-disubstituted benzene ring ( Table 1).
The 1 H nmR spectrum indicated signals assigned to a terminal vinyl group resonating at δ H 5.05 (1H, d, J= 1.8 Hz), 5.04 (1H, d, J= 1.8 Hz), and 5.69 (1H, ddd, J= 18.6, 12.0, 1.8 Hz), in addition to a singlet signal due to a quaternary methyl group resonating at 3.29 (3H, s). The 13 C nmR and DEPT spectra suggested that compound 1 possessed 19 carbon signals, which was categorized into indole-ring signals [(δ C 137.1 ppm (C, C-2), 107.5 (C, C-7), 128.3 (C, C-8), 118.3 (CH, C-9), 119.4 (CH, C-10), 121.4 (CH, C-11), 111.8 (CH, C-12), and 137.3 (C, C-13)], signals due to six methylenes, four methines, and one methyl carbons. Among them, two sp 3 methylenes (δ C 52.2 and 48.5 ppm), one sp 3 methine (δ C 55.6 ppm) attached to a nitrogen atom, and a methyl group (δ C 49.6 ppm) linked to the indole N-atom. The 2D spectra of compound 1 including the 1 H-1 H COSY, HSQC, and HMBC spectra showed great similarity to a previously isolated alkaloid known as antirhine) [24]. However, a deep comparison between both alkaloids revealed the absence of a methylene function in the case of 1 (CH 2 -6) and instead of the appearance of a methyl function. The location of the methyl function was deduced from the down-field absorptions in both 1 H and 13 C nmR and also from the correlation between these methyl protons and the carbons be linked to the nitrogen atom of the indole ring. From the comparison between the above results and literature data, compound 1 is greatly similar to antirhine [24], with some differences appearing when revising the HMBC spectrum with the correlation of the methyl group with C-2 and C-13. The relative stereochemistry of 1 was gleaned from the NOESY spectrum and by comparison of its chemical shift values with those of published data [24]. The cross-peak between H-15 and H-20 confirmed their cofacial orientation, and the absence of cross-peak between them and H-3 indicated the similarity of the stereochemistry of 1 with that of antirhine [24]. From the previous discussion, compound 1 and be identified as 6-nor-antirhine-N 1 -methyl (1) (Figure 1).

The Effect of Compound 2 on the Cell Cycle Distribution of Human Cancer Cells
Tracking cell cycle phases of the tumor cells were explored for anticancer effects. Therefore, the effect of compound 2 on the distribution of cell cycle phases was analyzed in MCF-7, HepG2, and HeLa cells using flow cytometry after treatment for 48 h. As illustrated in Figure 2, the proportion of MCF-7 and HepG2 cells in the G1 phase notably arrested increased by 25.7 ± 2.7% and 40.5 ± 2.0%, respectively, compared to the untreated cells. Meanwhile, the percentage of S phase in HeLa cells increased by 17.2 ± 1.8%. The percentage of cells in the G2/M phase significantly decreased by 59.2 ± 1.9% when HepG2 cells were treated with compound 2.

Assessing Cell Apoptosis with Annexin V-FITC
For differential assessment of the cells undergoing apoptosis (programmed cell death) versus cells dying via necrosis (non-programmed cell death) in MCF-7, HepG2, and HeLa cells, annexin V-FITC/PI staining coupled with flow cytometer was performed ( Figure 3). In the MCF-7, HepG2, and HeLa cells, a significant increase in apoptotic cell population was detected after treatment with 2, with 31.4 ± 0.2%, 29.2 ± 0.5%, and 34.9 ± 0.6%, respectively, compared to the cell control. In comparison to the control, compound 2 demonstrated a significant rise in the necrotic cell population after treating cervix cancer cells (HeLa) followed by hepatocellular carcinoma cells (HepG2) with 3.7 ± 0.2% and 2.5 ± 1.1%, respectively.

Figure 2.
Effect of compound 2 on cell cycle phases of MCF-7, HepG2 and HeLa cells. Cell cycle distribution was determined using DNA cytometry analysis after exposure to 2 for 48 h. Data are presented as the mean ± SD; n = 3; ** p < 0.01 and *** p < 0.001.

Assessing Cell Apoptosis with Annexin V-FITC.
For differential assessment of the cells undergoing apoptosis (programmed cell death) versus cells dying via necrosis (non-programmed cell death) in MCF-7, HepG2, and HeLa cells, annexin V-FITC/PI staining coupled with flow cytometer was performed ( Figure 3). In the MCF-7, HepG2, and HeLa cells, a significant increase in apoptotic cell population was detected after treatment with 2, with 31.4 ± 0.2%, 29.2 ± 0.5%, and 34.9 ± 0.6%, respectively, compared to the cell control. In comparison to the control, compound 2 demonstrated a significant rise in the necrotic cell population after treating cervix cancer cells (HeLa) followed by hepatocellular carcinoma cells (HepG2) with 3.7 ± 0.2% and 2.5 ± 1.1%, respectively. . Apoptosis/necrosis assessment for compound 2 against MCF-7, HepG2, and HeL subjected to previous treatment for 48 h, and apoptosis/necrosis quantified using flow cyto Data are presented as the mean ± SD; n = 3; ** p < 0.01 and *** p < 0.001.

Assessment of Autophagy
Aside from apoptosis, autophagy-mediated programmed cell death is a hot to science. The effect of compound 2 on the autophagy process in MCF-7, HepG2, and cells was evaluated using Cyto-ID autophagy detection dye and flow cytometry (F 4). Autophagic cell death was increased, triggered in MCF-7 and HeLa cells by 13.3 ± and 7.8 ± 0.1%, respectively, compared to the control percent. Additionally, the l percentage of the phagocytic pathway was observed with compound 2 on HepG2 ce 15.0 ± 0.1% compared to the control. . Apoptosis/necrosis assessment for compound 2 against MCF-7, HepG2, and HeLa cells subjected to previous treatment for 48 h, and apoptosis/necrosis quantified using flow cytometry. Data are presented as the mean ± SD; n = 3; ** p < 0.01 and *** p < 0.001.

Assessment of Autophagy
Aside from apoptosis, autophagy-mediated programmed cell death is a hot topic in science. The effect of compound 2 on the autophagy process in MCF-7, HepG2, and HeLa cells was evaluated using Cyto-ID autophagy detection dye and flow cytometry (Figure 4). Autophagic cell death was increased, triggered in MCF-7 and HeLa cells by 13.3 ± 0.1% and 7.8 ± 0.1%, respectively, compared to the control percent. Additionally, the lowest percentage of the phagocytic pathway was observed with compound 2 on HepG2 cells by 15.0 ± 0.1% compared to the control.

General
Instrument specifications, solvent sources, and grades, chromatographic separation materials, and reagents are previously reported [21].

Plant Material
The plant material was identified and collected as previously reported [23].

General
Instrument specifications, solvent sources, and grades, chromatographic separation materials, and reagents are previously reported [21].

Plant Material
The plant material was identified and collected as previously reported [23].

Cell Cycle Analysis
MCF-7, HepG2, and HeLa cells were treated with the pre-calculated IC 50 values of compound 2 for 48 h. Then, cells were harvested by trypsinization, twice washed with PBS (phosphate-buffered saline), fixed in ice-cold 60% ethanol at 40 • C, and re-washed in PBS. After that, cells were resuspended in 500 µL propidium iodide (PI) with RNase staining buffer, BD Pharmingen (Biosciences Inc, San Diego, CA, USA) and incubated for 30 min. Lastly, FACS analyses were executed utilizing the ACEA Novocyte™ flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA. For every sample, data of 12,000 cells were assembled and distribution of cell cycle phases were analyzed applying ACEA Novo Express™ software, ACEA Biosciences Inc., San Diego, CA, USA [27] 3.5.4. Apoptosis Analysis MCF-7, HepG2, and HeLa cells were treated with compound 2 for 48 h, trypsinized, and washed twice with PBS. Apoptosis assessment was carried out via the Annexin V-FITC/PI Apoptosis Detection Kit, BD Biosciences, San Diego, USA, as stated by the manufacturer. In brief, cells were resuspended in 0.5 mL of binding buffer then 5 µL of Annexin V-FITC and 5µL of PI (staining solution) were added for 15 min at room temperature in a dark place. Finally, the cells were applied to FACS analysis using ACEA Novocyte™ flow cytometer, ACEA Biosciences Inc., San Diego, CA, USA, within one hour following staining, cell cycle distribution is calculated using ACEA. Novo Express™ software (ACEA Biosciences Inc., San Diego, CA, USA) [28].

Autophagy Assay
Autophagic cell death was quantified using the Cyto-ID Autophagy Detection Kit (Abcam Inc., Cambridge Science Park, Cambridge, UK) to further explain the way by which cancer kills cells in response to compound 2 treatment. In brief, cells were treated to a predefined IC 50 of 2, for 48 h while being exposed to a drug-free medium (control group). Cells were collected and washed twice with PBS after treatment. Cells were stained with Cyto-ID Green and incubated at 37 • C for 30 min in the dark, according to the manufacturer's instructions. After staining, cells were injected and examined using ACEA Novocyte TM flowcytometry (ACEA Biosciences Inc., San Diego, CA, USA) [28].

Statistical Analysis
All data were analyzed using one-way analysis of variance (ANOVA.) Three replicates were used for each treatment. Differences between groups were considered significant at * p < 0.05, ** p < 0.01 and *** p < 0.001. Graphs were plotted using GraphPad Prism software, version 6.00 (GraphPad Software, La Jolla, CA, USA).